Published in: European Space Agency Special Report ESA SP 496, pp. 255-260.

FRONTIERS OF EXTREMOPHILIC MICROORGANISMS:
FROM LIFE ON THE EDGE TO ASTROBIOLOGY

Joseph Seckbach (1) and Julian Chela-Flores (2)

(1) Hebrew University of Jerusalem,
Home: P.O.B. 1132 Efrat 90435, Israel.
phone/fax: +972-2-993 1832 / E-mail: seckbach@huji.ac.il,

(2) The Abdus Salam International Center for Theoretical Physics,
Strada Costiera 11, PO Box 586; 34014 Trieste, Italy, and Instituto de Estudios Avanzados, Apartado 17606 Parque Central, Caracas 1015A, Venezuela.
phone +390-40-2240392 / fax: +390-40-22-42 41; e-mail: chelaf@ictp.trieste.it.

ABSTRACT

Extremophiles thrive on the edge of temperature, pH, pressure, hypersalinity, dryness, and desiccation. Such microorganisms may resemble the first living organisms which evolved on the early Earth. They may also serve as analogues of microbes on other worlds. Some celestial bodies may provide conditions for life, such as liquid water and other essential ingredients for microbial life. Consequently, life may exist elsewhere in the Solar System, even though the environments available may be more extreme than those on Earth. Wherever there is life we expect the presence of microbial life such as prokaryotic microorganisms. Prokaryotes have thrived on Earth at least since the period corresponding to 3.5 to 3.8 billion years before the present (Ga BP). They have dominated our biosphere during its first 2 Ga, even before the first eukaryote appeared, and have been the least affected through major climatic astronomical and geological events in the early Earth. Therefore, prokaryotes are the most likely candidates for a presumed biota on other worlds (although eukaryotes are also a possibility). The most promising worlds, which may harbor living microbes are Mars and the Jovian moon Europa.

INTRODUCTION

Life, as we know it, is based on carbon chemistry, moderated by liquid water in a given atmosphere. Life may occur throughout the Universe and a few planets may possess some earthlike conditions, which favor, or enable its inhabitability by microbes. There are hints that liquid water may be present on extraterrestrial environments, such as Mars or the Jovian satellite Europa (cf., the corresponding sections below). Furthermore, structures resembling nanobacteria have been observed in a Martian meteorite (AHL84001). It was discovered in Antarctica by McKay et al. (1996), leading the scientific community into a debate on its biological origin (Oro, 2000, 2001).
Prokaryotes were widespread on Earth as early as the period ranging from 3.5 to 3.8 Ga BP, while eukaryotes evolved 2 Ga later. Plants and animals appeared only during the past 600 million years. Microbial evolution led to diversification of life on Earth.
Unlike plants and animals, microbial life is abundant in a variety of ecosystems on our own planet. Much of astrobiology must rely on our knowledge of microbiology. Unfortunately, many questions still remain to be answered.
Furthermore, most bacteria have not yet been classified; the vast majority are living in ecosystems still waiting to be discovered. It is estimated that today less than 0.1% of all bacteria have been isolated and described.

ORIGIN OF THE FIRST CELLS

The first microorganisms have been heterotrophic and gained their energy from sunlight, reduced inorganic and organic compounds freely available before life originated.
Such energy sources included hydrogen gas, sulfide, sulfur, methane, ammonia, reduced iron, and hydrocarbons. These microbes obtain their energy by oxidizing these compounds in anaerobic conditions (absence of oxygen).
Microorganisms still exclusively utilize most of these energy sources chemosynthetically. The origin of life is considered to have occurred in warm or even hot areas at niches like hot springs, hydrothermal suboceanic vents and volcanic surroundings (Seckbach 1994/5; Davies, 1999).
Microorganisms detected deep under the surface of the Earth and oceans may be among the pioneers of life, who escaped the hazard of lethal UV radiation. Such environments protected microscopic life on the early Earth against the impact by celestial bodies.
The primordial atmosphere was characterized by high temperature and low pH; such atmosphere contained a high concentration of CO2, NH3 H2 and a low level of oxygen (see: Seckbach,1997, 1999, 2000a, 2000b; Seckbach et al. 1970; Siegel, 1999).
The CO2 biofixation on Earth started about 3.5 Ga BP, while the photosynthesis (carbon fixation with release of oxygen) took place during the same era. The first photosynthesizers were cyanobacteria, which left behind their traces as stromatolites and microfossils in several places all over the globe.
This bio-photoprocess is currently our main source of oxygen and energy. The primordial environment, mentioned above, is considered today as extreme and microbes had to tolerate, grow and thrive in those primeval conditions (Seckbach, 1994,1997; Davies, 1999).

EUKARYOGENESIS

It is generally accepted that prokaryotic cells evolved into eukaryotic (nucleated) unicellular microorganisms. There are two schools for explaining eukaryogenesis. The "classical" one is the autogenous path via compartmentalization in a "direct filiation" process (Seckbach, 1996; Jensen 1999, Nakamura, 1999). Additional aspects of extraterrestrial eukaryogenesis have recently been reported by Seckbach et al. (1998). The other theory for the evolution of the prokaryotes into the nucleated cell is the more popular (endo-) symbiosis concept.
According to the symbiotic theory, a free-living prokaryotic cell entered a host (prokaryote or eukaryote) larger cell. With time, and following the exchange of genomes, such symbiont (e.g. cyanobacterium, oxygenic eubacterium) turned into an organelle (i.e., chloroplast or/and mitochondrion, respectively). The origin of the eukaryotic cell nucleus may have originated from an archaean source (Horiike et al., 2001).
For more data on eukaryogenesis see, Seckbach (1996), Seckbach and Walsh (1999), Oren and Seckbach (2001), Ebringer and Kraj_ovi_ (1994). The relevance of eukaryogenesis to the search of extraterrestrial life has been discussed recently in the context of the Drake Equation (Chela-Flores, 2000).

THE EXTREMOPHILES

The special microorganisms which are able to colonize ecophysiological severe conditions are called "extremophiles". From our anthropocentric point of view these habitats are considered as "extreme" although by the microorganisms themselves these places are essentially 'oases'.
For other microbes such niches just lead to chaos and death. All three domains of life (Archaea, Bacteria, and Eukarya) are among the extremophiles (Oren and Seckbach, 2001; Roberts, 1999; Stetter 1998; Seckbach, 1997, 1999, 2000a, 2000b; Seckbach and Oren, 2000, 2001; Seckbach and Walsh, 1999). Various microbes on Earth developed a strategy to cope with a combination of extreme conditions found in their habitats, such as the cyanobacterium Chroococcidiopsis which survives a large variety of extreme conditions of dryness, acidity, salt and high as well as low temperatures. Cyanidium caldarium is a red thermoacidophilic alga which thrives in pure CO2 , at pH ranges of 0 to 4 and at maximum temperature level of 57ºC (Seckbach et al. 1970, Seckbach and Walsh, 1999; Walsh and Seckbach, 1999). Several archaean cells are acidophilic and hyperthemophilic such as Pyrolobus fumarri (9º to 113ºC) and Picrophilus oshimae or Thermoplasma acidophilum (~60ºC) thriving at very low levels of pH (Seckbach and Walsh, 1999; Seckbach and Oren, 2000; Stetter, 1998).
On the other hand, psychrophiles, the cold lovers, are abundant in a variety of places: the frigid zones of Antarctica, in ice samples removed from deep drills at Vostok Station, and in permafrost found in Siberia (Seckbach, 2000b, Seckbach and Oren, 2000b, 2001). The halophytes are microorganisms adapted to grow at saline and hypersaline environments like in the Dead Sea (Israel) and in the Great Salt Lake (Utah USA), or in salt crystals (Vreeland et al., 2000). The acidophiles thrive in very acidic environments, such as some prokaryotes (Thermoplasma or Pyrolobus), or eukaryotic algae, for instance the thermophilic Cyanidium caldarium (Seckbach, 1994), or Dunaliella acidophila (Pick, 1999); see also Oren and Seckbach (2001). Cyanobacteria may also occur in very alkaline surroundings (Boussiba et al., 2000; Oren and Seckbach, 2001). The bacterium Deinococcus radiodurans is highly resistant to UV radiation due to its multicellular walls, carotenoid pigmentation and a strong DNA repair mechanisms.
Another factor among the extremophiles is the dryness and desiccation. Dormant bacteria spores isolated from insects embedded in amber for 40 million years (Seckbach, 2000b), or from stomach of frozen mammoths, can be revived under suitable conditions.
Vreeland et al. (2000) claimed to isolate and grow a 250 million-year-old halotolerant bacterium from ancient salt crystals.
Further, it has been reported that the bacterium Streptococcus mitis, which has been left inside a TV camera aboard Surveyor 3 on the surface of the Moon for almost three years, could easily be revived upon retrieval of the camera and its transportation back to Earth by Apollo 12 (see Seckbach and Oren, 2000a). This revival occurred after these cells had been exposed there for a long time to vacuum, low temperature, UV radiation and without nutrients. Some bacteria, cyanobacteria, algae, fungi and protozoa survived following long periods of desiccation (Davis, 1972). Today these severe habitats are restricted to harbor extremophiles, which may resemble the first cells on early Earth and serve as analogues to representatives of extraterrestrial life. For additional information on extremophiles see Seckbach (1999, and 2000).

EXTREMOPHILES AS MODELS OF POSSIBLE SOLAR SYSTEM MICROORGANISMS

Three space missions have particularly clarified the possible sites where life should be searched in the form of microorganisms within the Solar System. They are firstly, the Voyager and Galileo missions to Jupiter and its satellites and, secondly, the Mars Global Surveyor (MGS). Several possible environments have emerged, where it is conceivable that microorganisms may have evolved and persisted, even up to the present day. Factors to keep in mind are:
A sufficient supply of organic matter and adequate sources of energy. The hypothesis that biological evolution, did take place elsewhere in the solar system triggered by means of exogenous sources, mainly by carbonaceous matter supplied by comets is increasingly receiving support. This hypothesis was originally formulated by Oro (2001), who discussed the role that cometary matter may have had on the formation of biochemical molecules on the early Earth. More recently has also developed by Greenberg (2001) in his studies of comet coma molecules, as well as by Owen and Bar-Nun (2000) in their work on planetary atmospheres. Those authors have developed earlier suggestions (Delsemme, 1992) that comets may have brought in a variety of volatile elements and compounds. At any rate for the principal candidates for hosting microorganisms, Europa and Mars, cometary delivery of organic matter is a viable possibility.
Life at hydrothermal vents has emerged as a valid alternative hypothesis for life's origin because those environments have the source of energy as well as protecting the incipient process of chemical evolution. The silicate interior of Europa can in principle provide active tectonic activity, although alternative sources for prebiotic evolution have been discussed recently (Phillips and Chyba, 2001). We cannot exclude that early Mars may have had widespread volcanic activity, as demonstrated by the large-scale features near the Martian equator, such as Olympus Mons.
A third key life-promoting factor remaining to be mentioned is whether there is liquid water in other environments of our solar system. We shall discuss this topic more fully in the last part of this work.

HAS THERE BEEN LIQUID WATER ON MARS?

The water inventory on Mars has been estimated from geomorphologic evidence. An obvious source of water ice lies on the north polar cap, which also contains dust ('dirty water') and carbon dioxide. The altimeter of the MGS has made a map of the northern polar ice cap. On the basis of these measurements it has been estimated that the diameter of this polar cap is 1,200 km and its maximum depth is 3 km. It is sufficient to cover the Martian surface to a depth of 10-30 m (or, equivalently about 4% of the total amount of water locked up in the ice of the Antarctic). An uncertainty is the unknown dust-to-water ratio. However, it is possible that liquid water existed in ancient times on the Martian surface, rather than simply water ice for reasons that will be seen below. We can estimate when liquid water flowed from a fairly accurate 'chronometer': the counting of craters on its surface. In the most remarkable outflow channels, which are in the northern hemisphere and drain into the Chryse basin, the crater count points towards an age of 3.5 Ga. The total amount of water estimated to have flowed along these channels is equivalent to covering the entire surface of Mars to a depth of 35 m.
Water activity is an indicator of warmer climate and thicker atmosphere; these conditions have led to naming this period the Martian "Eden". During this period, life may have originated on Mars at a time when the Sun was not as luminous as it is now. There must have been a greenhouse effect due to more abundant atmospheric carbon dioxide. A special comment must be reserved for the images of the MGS studied during the year 2000 which led the MGS camera team to infer that many of Mars's meteoritic craters were the site of lakes during part of their history (Malin and Edgett, 2000). According to their analysis the craters that have been considered contain accumulations of sedimentary rock that are several kilometers thick. The rock is divided into strata similar in color and thickness throughout Mars. The general distribution suggests that the sedimentation process was a global phenomenon, rather than the result of local events. It is generally accepted that Mars once had a thicker atmosphere than it does today, perhaps even comparable to Earth's. But where has the Martian atmosphere and microbial life gone? New evidence from NASA's MGS spacecraft supports a long held suspicion that much of the Red planet's atmosphere was simply "blown away by the (solar) wind". This is some of the strongest evidence to the present time for a Martian "Eden", when the whole planet was covered to a large extent by liquid water. This, in turn, is also strong support for the possibility that microorganisms may have originated on Mars, or at least from expelled terrestrial life, closer in time to the period of heavy bombardment during the Archean, which may have survived on favorable Martian conditions. There is now growing evidence that the physical and chemical surface properties of early Earth and Mars were very similar. Plenty of clues suggested that liquid water once flowed on Mars raising hope that life could have arisen there. It has been suggested that prior to 3.5 Ga the climate on Mars was wet and more temperate, allowing the presence of large quantities of water on its surface (Horneck, 2000). Under such conditions one would not eliminate the notion that early Mars has been favorable for life to emerge on its surface and subsurface as it did on the early Earth. McKay et al. (1996) reported the presence of polycyclic aromatic hydrocarbons (PAHs) and mineral grains of crystals of supposed biological origin in the Martian meteorite ALH84001, mentioned above. Furthermore, "nanofossils" shapes resembling were suggested on the basis of electron microscopy (McKay et al., 1996). Others point out that PAHs are present everywhere in the cosmos and even on Earth and hence they are not degradation products, which are specific of life. The same PAHs have been found in the Murchison meteorite. The evidence of small mineral grains of magnetite of supposedly biological origin may not be compelling. Finally, Oro (2000, 2001) proposed that the structures referred to as nanofossils may be just mineral formations of sizes from 10 to 100 times smaller than any independent terrestrial microorganisms with ribosomal and DNA structures. The final answer to this question may have to await confirmation after sample return missions retrieve pristine rocks in the future.
However, the question of life on Mars remains open, since organisms may have survived in very extreme conditions on that planet. Cyanobacteria and other microbes grow in Antarctica in severe cold, permafrost regions, dry environments, in dry deserts as well as in salt crystals or under high levels of pure CO2 (Seckbach et al., 1970); hence, these prokaryotes may serve as analogues for life on Mars. We still have to remember that MGS has uncovered some evidence for the presence of water on Mars, but final proof is still needed.

IS THERE LIQUID WATER ON EUROPA AND OTHER GALILEAN SATELLITES?

In 1976 the Voyager missions provided low resolution images of the surface of Europa. They showed a number of intersecting ridges and linae (cracks on the surface). The Galileo Mission has shown us that the central parts of some linae are of lower albedo than the surrounding terrain.
Some planetary scientists believe that these bright surface features may represent fresh ice that has come from below. The darker parts of the linae may represent silicate contamination also from below the surface, or alternatively ice that may have been darkened by other external, or internal factors.
Besides, we learnt that craters were not abundant, suggesting that Europa has been geologically active until a relatively recent date (or, alternatively, there may have been 'resurfacing' from liquid water from beneath the surface).
To sum up, Voyager supported the intuition that planetary scientists already had. Such confidence was based on two facts. Firstly, from Earth-bound spectroscopy we knew that Europa is covered with water ice. Secondly, the density of this satellite is not radically different from the Moon's density. From these remarks it follows that Europa is likely to have a silicate core. The Galileo mission has added much to our early insights.
An additional Europan feature is the presence of some form of 'ice-tectonics'. Some Galileo images suggest that part of the surface may be understood in terms of shifting plates of ice. From all the information gathered from Voyager and Galileo reasonable guesses have been put forward in the sense that there may be a substantial amount of liquid water between the silicate crust and the iced surface (Chyba, 2000). The trigger for the melting of the ice that we 'see' spectroscopically form the Earth could be tidal heating.
Magnetic field measurements from Galileo seem to confirm the existence of buried oceans, not only in Europa, but also in the large Galilean satellites: Ganymede and Callisto. But unlike Europa's ocean at a presumed depth of 10-100 km (underlain by a silicate crust with possibly volcanic activity), both Callisto and Ganymede would have much deeper oceans - up to 170 km and would be layered between ice above and below. Hence amongst the Galilean satellites Europa is the prime target for exploration for signs of living microorganisms (Phillips and Chyba, 2001).

IS EUROPA HARBORING LIFE?

Following the analyses of pictures taken of the surface of Europa, it is assumed that under its heavy ice sheaths this Jovian moon contains a liquid water ocean warmed up by volcanic sources. This water body may contain living organisms similar to those found in various places on Earth. For example, algae have been observed under ice layers in several places, and we know of algae which "paint" snow in various colors (Oren and Seckbach, 2001).
Prokaryotes and eukaryotes were observed in deep drills of Vostok Station in Antarctica (Karl et al. 1999; Oren and Seckbach, 2001). Perhaps one might also expect to find higher eukaryotic organisms in the extraterrestrial body like those tube worms living in hydrothermal vents at the depth of the oceans.
One could consider the appearance of microbial life only if the past conditions of this satellite were appropriate for the generation of biochemical compounds and their polymerization into more complex structures. Until landers and submersibles reach this Jovian moon, microbial life on Europa remains an open question.

CONCLUSIONS

Life is scattered all over the globe and microorganisms have been able to adapt to extreme environments. Understanding the ubiquity of terrestrial life suggests the possibility for life elsewhere in the universe; in particular, we have discussed some Solar System environments that may harbor extremophiles analogous to terrestrial ones. Further information on the extremophilic world has been recently published in books entitled:
Enigmatic Microorganisms and Life in Extreme Environments (published by Kluwer, 1999) and
Journey to Diverse Microbial Worlds: Adaptation to Exotic Environments (Kluwer, 2000),
both books have been edited by the senior author, see the following web sites:

http://www.wkap.nl/bookcc.htm/0-7923-5492-3
http://www.wkap.nl/bookcc.htm/0-7923-6020-6

ACKNOWLEDGEMENT

We thank ESA for financial support to attend the First European Workshop on Exo/Astrobiology, ESRIN, Frascati, Italy, 21-23rd May 2001. This chapter is also based on the senior authors' presentation at the XIIth rencontres de Blois, France. 25th June ­ 1st July 2000.

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